Title :

Assessing ER and Golgi N-glycosylation process using metabolic labeling in mammalian cultured cells

Author(s) :

Péanne, Romain [Auteur]
Vanbeselaere, Jorick [Auteur]
Vicogne, Dorothee [Auteur]
Mir, Anne-Marie [Auteur]
Biot, Christophe [Auteur]
Matthijs, Gert [Auteur]
Guerardel, Yann [Auteur]
Foulquier, Francois [Auteur]

Journal title :

Methods in cell biology

Volume number :

118

Pages :

157-176

Publication date :

2013

English keyword(s) :

Click Chemistry
Humans
Glycoprotein
Cells, Cultured
Glycosylation
Mannose
Endoplasmic Reticulum
N-Glycosylation
Sialic Acid
Golgi Apparatus
Congenital Disorders of Glycosylation
Staining and Labeling
Fibroblasts
Protein Processing, Post-Translational
Microscopy, Fluorescence
Fluorescent Dyes
Metabolic labeling

HAL domain(s) :

Chimie/Chimie théorique et/ou physique

English abstract : [en]

Modifications of N-glycosylation in disease states are common and illustrate the crucial requirement of glycosylation in human biology. Mainly based on glycan permethylation and the use of mass spectrometry analysis, we ...
Show more >Modifications of N-glycosylation in disease states are common and illustrate the crucial requirement of glycosylation in human biology. Mainly based on glycan permethylation and the use of mass spectrometry analysis, we can easily understand that many different methods to analyze the N-glycome have seen the day. While extremely powerful, these methods are mainly used to analyze qualitative variations of N-glycosylation of human serum proteins and do not necessarily reflect the glycosylation status of derived mammalian cultured cells. This chapter summarizes two methods that we are routinely using in our laboratory to assess the ER and Golgi N-glycosylation process. The proposed methodology allows pinpointing ER as well as Golgi glycosylation deficiencies in mammalian cultured cells. The first approach is based on direct metabolic labeling of cultured mammalian cells with [2-(3)H] mannose followed by sequential extraction and HPLC analysis of the purified oligosaccharides. The second one is based on the copper-catalyzed azide alkyne cycloaddition (CuAAC) strategy. We propose the use of alkyne-tagged sialic acid (SialNAl) to visualize the Golgi glycosylation efficiency. Their metabolic incorporation into newly synthesized glycoproteins can then be chemoselectively coupled to complementary azide-functionalized fluorophores, and visualized by using confocal laser scanning microscopy. To summarize, we present here a detailed description of our know-how in the field of ER and Golgi N-glycosylation.Show less >

Language :

Anglais

Administrative institution(s) :

CNRS
Université de Lille

Collections :

• Unité de Glycobiologie Structurale et Fonctionnelle (UGSF) - UMR 8576

Research team(s) :

Mécanismes moléculaires de la N-glycosylation et pathologies associées
Glycobiologie structurale des interactions hôtes-pathogènes

Submission date :

2020-02-12T15:12:11Z